The chemical moiety on the 5′ end of an RNA molecule influences its structure, stability, biochemical processing, transport, biological function and fate in a cell or organism. The chemical moieties commonly found at the 5′ end of RNA include triphosphates, monophosphates, hydroxyls, and cap nucleotides. The particular chemical moiety on the 5′ end provides important clues to the origin, processing, maturation and stability of the RNA. Characterization of this moiety in a newly identified RNA could even suggest a role for the RNA in the cell.
For example, bacterial mRNAs, small prokaryotic and eukaryotic ribosomal RNAs (e.g., 5S or 5.8S rRNAs), and transfer RNAs (tRNAs) typically have a 5′ triphosphate group.
Large ribosomal RNAs (e.g., 18S and 26S or 28S eukaryotic rRNA, or 16S and 23S prokaryotic rRNA), and eukaryotic or viral-encoded micro RNAs (miRNAs) typically have a 5′ monophosphate group. At least some initially-generated intron RNA molecules from pre-mRNA splicing reactions also have a 5′ phosphate group.
RNase A-degraded RNAs and some other endonucleolytically processed RNA molecules have a 5′ hydroxyl group.
Most eukaryotic cellular mRNAs and most eukaryotic viral mRNAs have a “cap” or “cap nucleotide” on their 5′ end (e.g., an “N7-methylguanosine” or “m7G” cap nucleoside that is joined via its 5′-carbon to a triphosphate group that, in turn, is joined to the 5′-carbon of the most 5′-nucleotide of the primary mRNA). Still further, some eukaryotic RNAs that are not translated into protein, referred to as “non-coding RNAs” or “ncRNAs,” have been described, and some of these are capped. Some capped ncRNAs also have a 3′ poly(A) tail, like most eukaryotic mRNAs. For example, Rinn, J L et al. (Cell 129: 1311-1323, 2007) described one capped and polyadenylated 2.2-kilobase ncRNA encoded in the HOXC region of human chromosome 12, termed “HOTAIR,” that has profound effects on expression of HOXD genes on chromosome 2. In addition, some other eukaryotic RNAs in a sample, such as small nuclear RNAs (“snRNAs”), and pre-miRNAs, can be capped.
The 5′ caps of eukaryotic cellular and viral mRNAs (and some other forms of RNA) play important roles in mRNA metabolism, and are required to varying degrees for processing and maturation of an mRNA transcript in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of the mRNA to protein. For example, the cap plays a pivotal role in the initiation of protein synthesis and in eukaryotic mRNA processing and stability in vivo. The cap provides resistance to 5′ exoribonuclease (XRN) activity and its absence results in rapid degradation of the mRNA (e.g., see Mol. Biol. Med. 5: 1-14, 1988; Cell 32: 681-694, 1983). Thus, mRNA prepared (e.g., in vitro) for introduction (e.g., via microinjection into oocytes or transfection into cells) and expression in eukaryotic cells should be capped.
Many eukaryotic viral RNAs are infectious only when capped, and when RNA molecules that are not capped (i.e., they are “uncapped”) are introduced into cells via transfection or microinjection, they are rapidly degraded by cellular RNases (e.g., see Krieg, and Melton, Nucleic Acids Res. 12: 7057, 1984; Drummond, et al. Nucleic Acids Res. 13: 7375, 1979).
The primary transcripts of many eukaryotic cellular genes and eukaryotic viral genes require processing to remove intervening sequences (introns) within the coding regions of these transcripts, and the benefits of the cap also extend to stabilization of such pre-mRNA. For example, it was shown that the presence of a cap on pre-mRNA enhanced in vivo splicing of pre-mRNA in yeast, but was not required for splicing, either in vivo or using in vitro yeast splicing systems (Fresco, L D and Buratowski, S, RNA 2: 584-596, 1996; Schwer, B et al., Nucleic Acids Res. 26: 2050-2057, 1998; Schwer, B and Shuman, S, RNA 2: 574-583, 1996). The enhancement of splicing was primarily due to the increased stability of the pre-mRNA since, in the absence of a cap, the pre-mRNA was rapidly degraded by 5′ exoribonuclease (Schwer, B, Nucleic Acids Res. 26: 2050-2057, 1998). Thus, it is also beneficial that transcripts synthesized for in vitro RNA splicing experiments are capped.
While capped mRNA remains in the cytoplasm after being exported from the nucleus, some other RNAs, such as some snRNAs have caps that are further methylated and then imported back into the nucleus, where they are involved in splicing of introns from pre-mRNA to generate mRNA exons (Mattaj, Cell 46: 905-911, 1986; Hamm et al., Cell 62: 569-577, 1990; Fischer, et al., J. Cell Biol. 113: 705-714, 1991). The splicing reaction generates spiced intron RNA that initially comprises RNA that has a 5′ monophosphate group.
Enzymes that modify the 5′ ends of RNA are useful tools for characterizing, studying, and manipulating various RNA molecules in vitro. For example, alkaline phosphatase (AP) (e.g., APEX™ alkaline phosphatase, EPICENTRE Technologies, Madison, Wis., USA; shrimp alkaline phosphatase, USB, Cleveland, Ohio; or Arctic alkaline phosphatase, New England Biolabs, MA) converts 5′ triphosphate groups (e.g., of uncapped primary RNA) and 5′ monophosphate groups (e.g., of rRNA) to 5′ hydroxyl groups, generating RNAs that have a 5′ hydroxyl group, but does not affect capped RNA. Nucleic acid pyrophosphatase (PPase) (e.g., tobacco acid pyrophosphatase (TAP)) cleaves triphosphate groups (e.g., of both capped and uncapped 5′-triphosphorylated RNAs) to synthesize RNAs that have a 5′ monophosphate group. A Dcp1/Dcp2 complex decapping enzyme (i.e., a “Dcp2-type” decapping enzyme) (e.g., yeast decapping enzyme, mammalian decapping enzyme, Arabidopsis thaliana decapping enzyme, vaccinia virus decapping enzyme, e.g., vaccinia virus decapping enzymes D9 or D10) converts capped RNA (e.g., m7G-capped RNA) to RNA that has a 5′ monophosphate group, but does not convert RNA that has a 5′ triphosphate group to RNA that has a 5′ monophosphate group. A capping enzyme (e.g., poxvirus capping enzyme, vaccinia virus capping enzyme, Saccharomyces cerevisiae capping enzyme, or SCRIPTCAP™ capping enzyme, EPICENTRE) converts RNA that has a 5′ triphosphate group or RNA that has a 5′ diphosphate group to capped RNA. Polynucleotide kinase (PNK) (e.g., T4 PNK) monophosphorylates hydroxyl groups on the 5′ ends of RNA molecules and removes monophosphate groups on the 3′ ends of RNA molecules (e.g., 3′ monophosphate groups generated from the action of RNase A). Further, 5′ exoribonuclease (XRN) (e.g., Saccharomyces cerevisiae Xrn I exoribonuclease, or TERMINATOR™ 5′-phosphate-dependent exonuclease, EPICENTRE) digests 5′-monophosphorylated RNA to mononucleotides, but generally does not digest RNA that has a 5′ triphosphate, 5′ cap, or 5′ hydroxyl group.
The reaction specificity of RNA ligase can also be a useful tool to discriminate between RNA molecules that have different 5′ end groups. This enzyme catalyzes phosphodiester bond formation specifically between a 5′ monophosphate group in a donor RNA and a 3′-hydroxyl group in an acceptor oligonucleotide (e.g., an RNA acceptor oligonucleotide). Thus, RNAs that have a monophosphate group on their 5′ ends, whether present in a sample or obtained by treatment (e.g., by treatment of 5′-triphosphorylated or 5′-capped RNA with TAP) are donor substrates for ligation to an acceptor nucleic acid that has a 3′ hydroxyl group using RNA ligase (e.g., T4 RNA ligase, EPICENTRE, or bacteriophage TS2126 RNA ligase, EPICENTRE). RNA molecules that have a 5′ triphosphate, diphosphate, hydroxyl or cap nucleotide do not function as donor molecules for RNA ligase. Thus, RNAs that have a hydroxyl group on their 5′ ends, whether present in a sample or obtained by treatment (e.g., treatment with AP) cannot serve as donor substrates for RNA ligase. Similarly, RNA molecules that contain a 3′-terminal blocking group (e.g., a 3′-phosphate group or a 3′-beta-methoxyphenylphosphate group) do not function as acceptor substrates for RNA ligase.
Numerous publications disclose use of alkaline phosphatase (AP), tobacco acid pyrophosphatase (TAP), and T4 RNA ligase to manipulate m7G-capped eukaryotic mRNAs (e.g., World Patent Applications WO0104286; and WO 2007/117039 A1; U.S. Pat. No. 5,597,713; Suzuki, Y et al., Gene 200: 149-156, 1997; Suzuki, Y and Sugano, S, Methods in Molecular Biology, 175: 143-153, 2001, ed. by Starkey, MP and Elaswarapu, R, Humana Press, Totowa, N.J.; Fromont-Racine, M et al., Nucleic Acids Res. 21: 1683-4, 1993; and in Maruyama, K and Sugano, S, Gene 138: 171-174, 1994). In those methods, total eukaryotic RNA or isolated polyadenylated RNA is first treated with AP, which converts RNA that has a 5′ triphosphate (e.g., uncapped primary RNA) and RNA that has a 5′ monophosphate to RNA that has a 5′ hydroxyl. Then, the sample is treated with TAP, which converts the 5′-capped eukaryotic mRNA to mRNA that has a 5′ monophosphate. The resulting 5′-monophosphorylated mRNA is then ligated to an acceptor oligonucleotide using T4 RNA ligase. The resulting “oligo-capped” mRNA is used for synthesis of first-strand cDNA, and double-stranded cDNA (e.g., to generate a full-length cDNA library and for identification of the 5′ ends of eukaryotic mRNA by sequencing or methods such as 5′ RACE).
In view of the importance of capped RNAs in gene expression and biological metabolism, there is currently great interest in studying and using the various types of capped RNAs for research, industrial, agricultural and medical purposes. Thus, what is needed in the art are improved methods for isolation, purification, production, and assay of capped RNA molecules in samples that also contain other uncapped RNA molecules.
Thus, what is needed are methods that enable selective removal of the uncapped RNAs under conditions wherein the capped RNAs are not removed. Enzymes can be useful tools for this purpose. However, prior to the present invention, no well characterized enzyme had been demonstrated in the art for selectively digesting the 5′ triphosphate of primary RNA, such as uncapped eukaryotic primary RNA or bacterial mRNA, to a 5′ monophosphate without also digesting capped eukaryotic mRNA. This is regrettable because an enzyme with this selective enzymatic activity could be used for isolating, purifying, manufacturing, or quantifying capped RNAs in a sample that also contains uncapped primary RNAs. Thus, what is needed in the art is a well-characterized RNA 5′ polyphosphatase enzyme, kits that contain said enzyme, and methods therefor.
What is needed in the art are RNA 5′ polyphosphatase compositions that are capable of converting a 5′ triphosphate group of a primary RNA transcript to a 5′ monophosphate group, and methods for using said RNA polyphosphatase enzyme compositions in order to selectively convert undesired uncapped primary RNAs that have a 5′ triphosphate group to RNAs that have a 5′ monophosphate group without also converting desired capped RNAs to RNAs that have a 5′ monophosphate group.
What is further needed are methods, compositions, and kits that employ one or more other enzymes, in combination with and in addition to an RNA 5′ polyphosphatase enzyme composition, in order to selectively remove both RNAs that have a 5′ monophosphate group in a sample, as well as the RNAs that have a 5′ monophosphate group generated as a product of the RNA 5′ polyphosphatase enzymatic reaction, thereby removing those RNAs from the capped RNAs present in the sample (e.g., for preparation of compositions that consist of only capped RNA molecules, e.g., for expression in eukaryotic cells, e.g., in oocytes or somatic cells, e.g., for research and therapeutic applications).
Still further, enzymes that are capable of removing phosphate groups (e.g., phosphatases and pyrophosphatases) are widely known in the art and have been widely used as signal-amplifying substances for detection of biomolecules for research, molecular diagnostics, immunodiagnostics, and other applications. For example, such phosphate-removing enzymes have been widely used for making conjugates with small molecules like biotin or digoxigenin and with nucleic acids or proteins (e.g., streptavidin, protein A, or primary or secondary antibodies) for use as signal-amplifying substances for sensitive detection of nucleic acids, proteins, and other analytes. One widely used phosphate-removing enzyme is alkaline phosphatase derived from calf intestine or bacteria. However, since the signal-amplifying enzymes used in the art are active as homodimers and require divalent metal cations for catalysis, these enzymes may be undesirable for certain assays because their subunits could dissociate, resulting in low assay sensitivity. Also, because the signal-amplifying enzymes in the art require divalent metal cations, their use in some assays is difficult or impossible, or necessitates additional assay steps, which is inconvenient. Thus, what is needed in the art are single-subunit enzymes with phosphate-removing enzymatic activities that are active in the absence of divalent metal ions for use as signal-amplifying substances for sensitive detection of nucleic acids, proteins, or other analytes. What is needed are such single-subunit enzymes that can be used to make conjugates with affinity binding molecules for use as signal-amplifying substances.
Also, since the signal-amplifying enzymes used in the art are active as homodimers, it is more difficult to genetically engineer and make fusion proteins consisting of the signal-amplifying enzyme and a proteinaceous affinity binding molecule (e.g., streptavidin, a single-chain artificial antibody, or protein A). Thus, what is further needed in the art are single-subunit enzymes that can be used to genetically engineer fusion proteins consisting of the signal-amplifying enzyme and a protein affinity binding molecule for use as signal-amplifying substances.